Project Summary Parkinson?s disease (PD) is characterized by motor abnormalities primarily caused by loss of midbrain dopamine (DA) cells, which largely modulate the striatal neurons. DA depletion is thus associated with altered function of striatal projection neurons (SPNs). SPN dysregulation is evidenced by significant morphological and physiological changes, as shown in numerous in vitro/ex-vivo studies. Furthermore, our studies in primate models and patients have revealed pathological hyperactivity of SPNs. A key contributor to this change is likely the excitatory glutamate signaling. To address the glutamate role, we have tested the effects of intrastriatal delivery of selective antagonists in parkinsonian primates, and showed that blocking the NMDAR transmission controls the typically altered SPN responses to DA replacement. Thus, these data support that reducing glutamate signaling on SPNs may have therapeutic effects in PD. However, pharmacotherapy is of limited use because of widespread action in the brain of available drugs that can cause significant off-target effects. Here, we propose to explore a new therapeutic strategy based on silencing NMDAR subunit genes in the striatum, specifically GluN2 subunits, to affect synaptic function. This strategy that reduces glutamate signals on SPNs will produce beneficial motor effects, as shown by local NMDAR blockers. Moreover, gene silencing will induce more persistent tuning of the SPN activity that will likely result in additional benefits, including improvement of both basal parkinsonism and DA responses. To investigate this gene therapy, we will use viral vector injections in the striatum to induce small hairpin RNA expression that interferes specifically with the target gene, i.e. GluN2A, B or D subunit gene. In this initial phase, our plan is to test gene suppression in the striatum of rats with nigrostriatal DA lesion and chronic exposure to L-Dopa. We propose two specific aims. In the first aim, we will analyze motor effects to identify the best subunit target, and then examine long-term benefits and potential adverse effects, including mutagenic and immunogenic changes. In the second aim, we will confirm effects with a highly controlled study, and validate the target with physiological effects of gene silencing. This project employs experimental approaches to address the main points of a new therapeutic development. These approaches include the use of novel viral vectors, established animal models, and specific behavioral, histological/molecular and electrophysiological analyses. The main goals of this initial study are: (I) to demonstrate in vivo efficacy and target engagement, and (II) to generate pivotal data that stimulate continuation of this research. The ultimate goal of this project is to develop a new therapy to improve motor function in PD.